Many members of the Bunyaviridae family of segmented negative sense RNA viruses pose a serious threat to human health, and in recognition of this fact, several of these viruses are described by the CDC as category A, B or C priority pathogens. Bunyamwera virus (BUNV) is the prototype of both the Orthobunyavirus genus and also the Bunyaviridae family, and it provides a model for studying the molecular and cellular biology of the serious human pathogens that reside within this family of viruses. Using BUNV, our long-term objective is to define the roles of cis- and trans- acting factors in controlling mRNA transcription and RNA replication, and further to understand how these factors coordinate relative expression levels of the genes within the segmented BUNV genome. This understanding will both aid in the design of preventative or therapeutic strategies that selectively target and interfere with bunyavirus RNA synthesis processes without adversely affecting host cell function, and will also provide a rational strategy for design of growth-attenuated bunyaviruses as candidates for development of live vaccines. The segmented arrangement of the bunyavirus genome dictates that relative gene expression will depend on the ability of each segment to perform both transcription to generate more mRNAs, and also RNA replication to generate more transcriptionally active genome templates. To allow analysis of bunyavirus gene expression, we have developed a novel RNA synthesis system that allows direct detection and analysis of the products of both RNA replication and mRNA transcription generated by synthetic cDNA-derived BUNV segments. The three specific aims listed in this application have been designed to exploit this novel system to generate a platform of necessary information that will guide future investigations. Aim 1) to determine the role of nucleotides within the non-coding regions of the BUNV genome in signaling transcription and replication; Aim 2) to identify cis-acting sequences involved in BUNV transcription termination; Aim 3) Investigation of the role of the BUNV N protein in control of BUNV RNA synthesis. [unreadable] [unreadable]